Download Direct redox modulation of p53 protein: potential sources of redox

Gene Therapy and Molecular Biology Vol 4, page 119
Gene Ther Mol Biol Vol 4, 119-132. December 1999.
Direct redox modulation of p53 protein: potential
sources of redox control and potential outcomes
Review Article
Hsiao-Huei Wu1 , Mark Sherman2 , Yate-Ching Yuan3 , and Jamil Momand1, *
1
Dept. of Cell and Tumor Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte CA 910103000
2
Dept. of Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte CA 91010-3000
3
Dept. of Biomedical Informatics, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte CA 910103000
______________________________________________________________________________________________________
*
Correspondence: (Present address) Jamil Momand, Ph.D., Department of Chemistry, California State University at Los Angeles, 5151
State University Drive, Los Angeles CA 90032. Tel: 323-343-2361; Fax: 323-343-6490; E-mail: [email protected]
Abbreviations: ROI, reactive oxygen intermediates; Ref-1, Redox factor-1; DTT, dithiothreitol; ESR, electron spin resonance; PDTC,
pyrrolidine dithiocarbamate; GSH, glutathione; SOD, superoxide dismutase; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine
Key Words: oxidation, cysteine, pyrrolidine dithiocarbamate, stressor, sulfhydryl
Received: 29 April 1999; accepted: 17 May 1999
Summary
Appropriate response to environmental stressors is essential for life. Many stressors, such as UV light, ionizing
radiation, reactive oxygen intermediates (ROI), heat shock and hypoxia alter the redox potential of the cell. Recently, it
has been shown that some of these stressors promote direct oxidation of specific protein cysteine residues resulting in
either up-regulation or down-regulation of protein activity in the cytosol. In higher eukaryotes, the p53 tumor
suppressor gene is a central component of stress response and its activation results in either cell cycle arrest or
apoptosis. In cultured cells, p53 appears to become activated by some stressors (hydrogen peroxide, heat) predicted to
directly increase cellular redox potential. However, in vitro studies indicate that p53 protein oxidation inhibits its
ability to bind its consensus sequence DNA. If p53 is unable to bind consensus sequence DNA, p53 is predicted to be
incapable of activating the p21WAF1/CIP1 gene, responsible for mediating G1 cell cycle arrest. Two proteins previously
shown to reduce oxidized cytoplasmic proteins, Redox factor-1 and thioredoxin reductase, have been shown to play
important roles in maintaining p53 activity, suggesting that they may be responsible for keeping p53 in the reduced
state inside the cell. Analysis of the p53 crystal structure revealed several well-conserved cysteine residues exposed on
the protein surface that may be susceptible to oxidation. Based on this analysis we predict that cysteine residues 124,
176, 182, 242 and 277 are primary candidates for redox regulation. In this communication, we review the data
demonstrating p53 regulation by direct alteration of p53 cysteine residue oxidation, propose a testable mechanism by
which p53 oxidation may occur, and discuss the possible implications of p53 oxidation on cell growth control and DNA
repair.
Once activated, the intracellular p53 protein level increases
and p53 binds, in sequence-specific fashion, to certain DNA
promoters which, in turn, leads to activation of genes that
mediate cell cycle arrest (El-Deiry et al., 1993; Chin et al.,
1997; Hermeking et al., 1997; Bunz et al., 1998) or apoptosis
(Miyashta and Reed, 1995). One of the p53 responsive
genes that appears necessary for mediating G1 arrest in
several cell types is p21WAF1/CIP1 , a cyclin-dependent kinase
inhibitor (El-Deiry et al., 1993; Harper et al., 1993).
Activation of p53 is complex and inhibition of this process
can lead to loss of cell growth control.
I. Introduction
The p53 tumor suppressor gene is one of the most
frequently mutated genes in human cancers (Baker et al.,
1989; Nigro et al., 1989; Hainaut et al., 1998). It is a cell
cycle checkpoint gene responsible for committing
mammalian cells to a growth arrest phenotype or apoptosis
in response to genotoxic and non-genotoxic stressors
(Levine, 1997; Giaccia and Kastan, 1998). The p53 gene
encodes a transcription factor that is synthesized in a latent
form and can be activated by a wide range cell stressors.
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be oxidized at cysteine residues in cells and the effect of
oxidation on their activities. For illustrative purposes, a few
of these will be discussed.
Three types of chemical oxidation have been identified
on protein cysteine sulfhydryl groups in cells. The first type
of oxidation is intramolecular disulfide bond formation.
This was shown to occur on two bacterial proteins, OxyR
and Hsp33 (Zheng et al., 1998; Jakob et al., 1999).
Treatment with hydrogen peroxide or heat leads to the
formation of intramolecular disulfide bonds and results in
the activation of the protein as a transcription factor, in the
case of OxyR, or a chaperone protein, in the case of Hsp33.
Oxidized OxyR transactivates a panel of genes responsible
for protecting the organism from hydrogen peroxide
poisoning
including
hydroperoxidase
I,
alkyl
hydroperoxidase reductase and glutathione reductase
(Jamieson and Storz, 1997). Oxidized Hsp33 protects
enzymes from denaturing during heat stress or hydrogen
peroxide treatment (Jakob et al., 1999).
Activation of p53 is thought to take place at both the
translational and post-translational level. Most recent work
has concentrated on understanding the post-translational
events that lead to p53 activation. The exact activation
pathway is highly dependent on the type of stressor applied.
Each class of stressors appears to result in a unique pattern
of p53 protein phosphorylation and acetylation to achieve
p53-mediated transcription of appropriate downstream
targets (Giaccia and Kastan, 1998). Concomitant with these
modifications p53 protein levels increase. Another potential
post-translational modification system less extensively
explored is direct p53 redox regulation. Protein redox
alterations in response to environmental agents, was
proposed to occur more than four decades ago (Barron,
1951), but it is only within the past 5 years that, with the
advent of new techniques, solid evidence has accumulated
indicating that redox changes can occur on cytoplasmic
proteins in vivo (Åslund and Beckwith, 1999). Table 1 lists
a partial set of cytoplasmic proteins that have been shown to
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Gene Therapy and Molecular Biology Vol 4, page 121
Figure 1. Schematic diagram of three basic p53 protein functional domains in human p53. Primary protein sequence of p53.
The numbered serine (S) and lysine (K) residues are sites of phosphorylation and acetylation respectively. All cysteine residue positions are
shown.
modifications, thus far, have been shown to occur in the Nand C-terminal domains. The N-terminal 42 amino acid
residues is required for p53-mediated transactivation. For
p53 to mediate transcription, this domain must bind
hTAFII31 and hTAFII70 accessory transcription factors that
form part of the TATA box binding protein complex TFIID.
Six serine residues within the N-terminal domain can be
phosphorylated. Phosphorylation at Ser 15 is required, in
some instances, to upregulate p53 protein levels (Shieh et al.,
1997). The C-terminal domain (301-393) includes the
tetramerization domain, the nuclear localization sequence
and a nuclear export sequence. There are four serine
residues regulated by phosphorylation and two lysine
residues regulated by acetylation in the C-terminal domain.
The central domain of p53 (residues 100-300) can bind DNA
in a sequence-dependent manner at two palindromic halfsites with the sequence 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3'.
Mutations in p53 observed in human tumors and
malignancies almost always map to this central DNA
binding domain. Proteins with missense mutations in this
domain are usually incapable of binding p53 consensus
sequence containing DNA elements. All conserved cysteine
residues are observed in this domain and, in some cases, Cys
residues are mutated in cancers. The frequency of the
mutations is fairly low and therefore, cysteine residues are
not considered as mutation hot spots (Sun and Oberley,
1996). Direct redox changes on p53 itself would likely
affect one or more of these cysteine residues. Given their
close proximity to the DNA binding domain it is likely that
oxidation of some critical cysteine residues will affect
sequence-specific binding activity of p53.
The second type of chemical oxidation involves the
oxidation of a metal-sulfur center active site. The bacterial
transcription factor SoxR falls into this category (Ding et al.,
1996). Like OxyR, SoxR activates genes responsible for
bacterial oxidation defense. Target genes upregulated by
SoxR include Mn-containing superoxide dismutase, glucose6-phosphate dehydrogenase and the DNA repair enzyme
endonuclease IV. Aside from intramolecular disulfide bond
formation and oxidation of metal sulfur centers, a third type
of chemical oxidation that occurs in proteins is a disulfide
bond formed between a protein cysteine residue and a small
molecular weight thiol molecule. Some enzymes have been
shown to form a disulfide bond with glutathione (Thomas et
al., 1994; Cabiscol and Levine, 1996) and cysteine (Sato et
al., 1996). Recently, it was shown that p53 is oxidized in
cultured cells in the presence of a metal chelator/oxidant,
called pyrrolidine dithiocarbamate (PDTC) (Wu and
Momand, 1998).
Oxidation of p53 correlated with inhibition of its
transactivation activity. The chemical nature of the p53
cysteine residue oxidation is not known nor, aside from
PDTC, are the types of other stressors that lead to p53
oxidation. In this review, we hope to shed light on the
possible reactive cysteine residues within p53 and the role
direct redox regulation plays in modulating this protein's
function.
To understand how oxidation might affect p53 function
it is important to review, briefly, the location of the different
functional domains of p53 in relation to the p53 cysteine
residues (for extensive reviews on this subject see Gottlieb,
1996; Greenblatt, 1994). As shown in Fig. 1, the p53
protein can be divided roughly into three distinct domains
based on function.
All regulatory post-translational
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increase intracellular hydrogen peroxide or hydroxyl radical
concentration can lead to elevation in p53 protein levels, p53
nuclear accumulation, p53-dependent cell growth arrest, and
p53-dependent cell death in some cell types.
II. p53 activity is upregulated in
response to agents that increase
intracellular reactive oxygen intermediates
Protein disulfide formation can occur in the cytosol
when intracellular reactive oxygen intermediates (ROI) are
created (Halliwell and Gutteridge, 1989). ROI have long
been thought to be part of the multitude of small intracellular
molecules that signal specific transduction pathways within
the cell. The three common types of intracellular ROI
postulated to be important for modulating protein redox
levels are hydrogen peroxide, hydroxyl radicals and
superoxide. Specific enzymes have evolved to rid the cell of
hydrogen peroxide and superoxide and these enzymes are, in
fact, regulated in response to these molecules. Intracellular
peroxides are generated at sites of inflammation by secretion
of hydrogen peroxide by neutrophils (Vile et al., 1998).
Hydroxyl radicals are generated in cells in response to UV
light, ionizing radiation and free metals. Interestingly,
doxorubicin, a common chemotherapeutic agent used in the
treatment of tumors, also appears to generate hydroxyl
radicals, that may contribute to its anti-neoplastic properties
(Doroshaw, 1986).
Both genetic and cell biology studies suggest that p53
can be activated by stimuli that also result in intracellular
ROI production. Solar UV radiation leads to cellular p53
protein elevation and agents that scavenge hydroxyl radicals
prevent p53 protein elevation (Vile, 1997). In cultured
normal human fibroblasts, treatment with Cd, a metal known
to catalyze the formation of hydroxyl radicals, or hydrogen
peroxide leads to accumulation of p53 in the nucleus, a sign
of p53 activation in some cells (Sugano et al., 1995; Uberti
et al., 1999). In one study, normal human fibroblasts treated
with a sublethal dose of hydrogen peroxide underwent long
term growth arrest, suggestive of p53 activation (Chen et al.,
1998). In IMR-90 fetal lung cells, p53 and p21WAF1/CIP1
protein levels were transiently elevated in response to
hydrogen peroxide (Chen et al., 1998). Hydrogen peroxidemediated upregulation of p53 protein was inhibited by the
iron chelator deferoxamine, suggesting that intracellular
hydroxyl radical formation, perhaps generated by Fentontype chemical reactions, is an important component of this
signaling pathway. When the viral oncoprotein E6, a p53inhibiting protein, was expressed in IMR-90 cells, H2O2
treatment failed to upregulate p53 levels or to induce G1
arrest and there was a diminution in the level of p21WAF1/CIP1
increase. These studies suggest that H2O2 and most likely
hydroxyl radicals are important intracellular molecules that
lead to p53 activation and cell cycle arrest.
Increases in intracellular ROI levels can also lead to
p53-dependent programmed cell death. Using murine
embryo fibroblasts cells derived from p53 -/- mice, it was
shown that hydrogen peroxide leads to p53-dependent cell
death (Yin et al., 1998).
Similarly, normal human
fibroblasts engineered to express E6 failed to undergo
programmed cell death while normal human fibroblasts
without E6 underwent programmed cell death in response to
hydrogen peroxide suggesting that p53 is required for cell
death (Yin et al., 1999). In summary, agents known to
III. Studies on redox regulation of p53
in vitro
One of the possible mechanisms of p53 activation by
ROI is direct oxidation of the p53 protein. Oxidation may
activate p53 for cell cycle arrest and apoptosis. However, to
date, all evidence from p53 oxidation studies conducted in
vitro indicates that sequence specific DNA binding is
inhibited by p53 oxidation (Hupp et al., 1992; Hainaut and
Milner, 1993; Delphin et al., 1994; Sun and Oberley, 1996).
Evidence that p53 cysteine residue oxidation can prevent
p53 from properly binding its DNA consensus sequence
comes from the fact that: (i) high concentrations of
dithiothreitol (DTT) are required (0.1-10 mM) to allow
recombinant p53 or p53 in nuclear extracts to bind DNA; (ii)
treatment of purified recombinant p53 with the thiol
alkylating agent N-ethyl maleimide (Rainwater et al., 1995)
or in vitro translated p53 with diamine (Hainaut and Milner,
1993) prevents p53 from binding to its DNA consensus
sequence. Thus, it appears that maintenance of p53 cysteine
residues in the reduced state is necessary for optimal p53
consensus sequence-dependent DNA binding.
Another assay to test whether ROI may modulate p53
activity is the p53-transactivation assay. In this assay, a
plasmid expressing p53 is cotransfected with a plasmid
encoding a p53-responsive element placed upstream of a
gene that codes for a transcription reporter. In one report it
was demonstrated, using this assay, that H2O2 inhibited p53mediated transactivation (Parks et al., 1997) consistent with
the data demonstrating that oxidized p53 fails to bind DNA
in vitro. This result, at the outset, appears inconsistent with
data demonstrating that H2O2 treatment correlates with an
increase in p53 protein and transactivation of the p21WAF1/CIP1
gene (Chen et al., 1998). Indeed, nuclear extracts from cells
treated with H2O2 contain higher levels of p53 sequence
specific DNA binding activity than untreated cells
(Verhaegh et al., 1997).
A possible explanation for this apparent contradiction
is that H2O2 treatment of cells may directly oxidize p53 and,
in addition, may lead to higher levels of p53 protein. The
oxidized p53 is expected to be incapable of binding p53dependent effector genes in vivo. However, it must be kept
in mind that nuclear extracts derived from H 2O2-treated cells
often include DTT in the DNA-binding buffer. In this case,
the high levels of oxidized p53 may be rapidly converted to
a reduced form that can bind consensus sequence containing
DNA. Rapid reduction of p53 by DTT may explain why the
p53 DNA binding capacity appears higher in H2O2-treated
cells. Notwithstanding this argument, one must still explain
the apparent p53-dependent upregulation of p21WAF1/CIP1
observed in cultured cells after H 2O2 treatment. It is possible
that H 2O2 treatment immediately leads to high levels of
transcriptionally inactive p53. After initial oxidation of p53,
the oxidized cysteine residues on p53 may be reduced by
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specific enzymes that are also activated by H2O2. Upon
reduction, p53 may then upregulate p21WAF1/CIP1 . If this
prediction is correct, one would expect that H 2O2 treatment
would lead to delayed activation of p21WAF1/CIP1 . Such a
prediction is consistent with the fact that H 2O2 treatment of
cells leads to an increase in p53 protein at 1.5 h posttreatment and to p21WAF1/CIP1 increase at 18 h post-treatment
(Chen et al., 1998).
If this scenario is correct then the molecules required to
maintain p53 in a reduced state may, in some instances, be
limiting within the cell. This conjecture is supported, to
some extent, by the fact that the DNA-binding activity of
recombinant mouse p53 in freshly prepared nuclear extracts
from baculovirus-infected insect cells is stimulated by
treatment with DTT (Delphin et al., 1994). It is possible that
p53 protein is overexpressed in insect cells relative to the
reducing molecules needed to keep the p53 in the reduced
state. This model raises the question of whether enzymes
involved in reducing oxidized protein cysteine residues
affect p53 activity.
p53 (p53 lacking its 30 C-terminal amino acid residues) is
used in the DNA-binding assay.
This is somewhat
unexpected because there are no cysteine residues within this
C-terminal region of p53. However, the C-terminus does
appear to normally negatively regulate the sequence specific
DNA binding function of p53 (Hupp et al., 1992, 1993;
Hupp and Lane, 1994). Ref-1 and p53 do not form a stable
complex regardless of whether DNA is present. It is
possible, then, that Ref-1 transiently associates within the
terminal 30 amino acid residues of p53 and reduces oxidized
p53 cysteine residues within the central-DNA binding
domain of p53. Importantly, Ref-1 was observed to increase
p53 transactivation activity in transient expression assays.
When the Ref-1 endonuclease domain was removed, Ref-1's
ability to stimulate p53 DNA binding activity was severely
inhibited but not completely abolished. The data indicate
that Ref-1 may stimulate p53 DNA binding activity through
both, a non-redox and a redox mechanism. Genetic studies
using REF1 +/- and REF1 +/+ mice in appropriate genetic
backgrounds suggest that p53 activation in response to UV
irradiation is dependent on Ref-1 (Meira et al., 1997).
IV. Enzymes that may be responsible
for maintaining p53 in the reduced state
VI. Thioredoxin reductase
Thioredoxin reductase is another enzyme that may be
responsible for reducing p53 cysteine residues, either
directly or indirectly. Thioredoxin reductase is a protein
disulfide reductase that catalyzes NADPH-dependent
reduction of the active site disulfide in oxidized thioredoxin,
a small protein (12-14 kD), to a vicinal dithiol (Arner et al.,
1999). The requirement of thioredoxin reductase for human
p53 activity was identified in a genetic complementation
study in the yeast strain Schizosaccharomyces pombe (Casso
and Beach, 1996). In this yeast strain, ectopically expressed
human p53 causes growth arrest (Bischoff et al., 1992).
Casso and Beach (1996) found that a mutation in a yeast
homologue of the human thioredoxin reductase gene (trr1)
rescued p53-dependent growth arrest.
p53-mediated
transcription was also downregulated by this mutant allele of
trr1. Mutant trr1 required O 2 for its inhibitory effect on
p53-mediated growth arrest. A strain of S. pombe lacking
trr1 acted in a similar manner to the strain expressing the
mutant trr1, suggesting that the original mutant trr1 acted as
a dominant negative allele. The requirement for thioredoxin
reductase in order to maintain the transcriptional activity of
p53 was also demonstrated in the evolutionarily distant to S.
pombe yeast strain Saccharomyces cerevisiae (Pearson and
Merrill, 1998). These results suggested that either p53 itself,
or a protein required for p53 function, is susceptible to
disulfide bond formation. Once the disulfide bond is formed
p53-mediated transactivation is abrogated. Thioredoxin
reductase is required to reduce the disulfide bond and restore
p53 function.
It is possible that some redox reactions are controlled
by subcellular localization of redox-sensitive factors.
Interestingly, translocation of cytoplasmic thioredoxin to the
nuclear compartment of mammalian cells was recently
demonstrated (Hirota et al., 1997). Furthermore, thioredoxin
and Ref-1 can form a complex in vitro and in vivo . The
Redox control of p53 may be a chemical or enzymatic
process. No consistent data has emerged to indicate the
presence of a protein oxidizing enzyme in the cytoplasm.
However, several enzymes appear to participate in reducing
cytoplasmic protein disulfide linkages (Thomas et al., 1995;
Rietsch and Beckwith, 1998). Thus, it is possible that redox
regulation of p53 occurs by chemical oxidation and
enzymatic reduction. Although no redox enzyme can be
excluded from involvement, evidence to date indicates that
there are two candidate enzymes responsible for maintaining
p53 in a reduced state in eukaryotic cells: Ref-1 and
thioredoxin reductase.
V. Ref-1
Redox factor-1, or Ref-1, was characterized as an
activity from HeLa cell nuclear extracts that increased
recombinant p53 binding to a p53 consensus sequence
(Jayaraman et al., 1997). Ref-1 was previously shown to
increase the activity of Fos-Jun heterodimer binding to DNA
in a manner that depended on DTT (Xanthoudakis and
Curran, 1992). Similar to p53, Fos and Jun are redoxsensitive transcription factors that directly bind DNA and
upregulate transcription of genes involved in cell cycle
progression (Abate et al., 1990). Ref-1 also possesses class
II hydrolytic apurinic/apyrimidinic (A/P) endonuclease
activity (Demple et al., 1991; Robson and Hickson, 1991).
Because of this latter function, Ref-1 has also been assigned
other names such as APE, APEX, and HAP-1. The redox
regulation portion of Ref-1 and the endonuclease activity of
Ref-1 lie within separate domains of the protein.
In the presence of DTT, Ref-1 stimulates consensus
DNA binding of full-length p53 but this stimulatory activity
is severely inhibited when a C-terminally truncated form of
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precise mechanism and the role of thioredoxin, Ref-1 and
thioredoxin reductase in the regulation of p53 activity in
mammalian cells is unknown but offers a potentially fertile
environment for further experimental exploration.
If p53 forms an intramolecular disulfide bond it is
important to consider the relative orientation of the two
cysteine residues in question. One measure of the degree of
cysteine residue side chain movement required for disulfide
bond formation is to compare the χ1 dihedral angles of p53
Cys176 and Cys242 to the χ1 dihedral angles of disulfides in
known protein structures. In early work, Richardson (1981)
measured the χ1 dihedral angles of 70 protein disulfide
bonds and found that the χ1 angles tended to, but did not
exclusively, cluster at -60° (±20°), +180 (±20°) and +60°
(±20°). The relative frequency of these angles in protein
disulfides was -60°>180°>+60°. In p53, the measured χ1
angle of Cys176 is +74° while that of Cys242 is -90°. Thus,
the χ1 angle of Cys176 falls within the least frequent cluster
of observed disulfide dihedral angles (+60° (±20°)) while the
χ1 of Cys242 does not fall into any particular class. If the
zinc atom is removed, the cysteine residue side chains may
undergo slight changes to form more favorable dihedral
angles for disulfide bond formation.
A third factor influencing the likelihood of disulfide
bond formation is the polarity of the amino acid residues
near the cysteine residue (Snyder et al., 1981). Residues that
are positively charged and in close proximity to a cysteine
residue could attract negatively charged disulfides, such as
glutathione disulfide, to the reactive cysteine residue on p53.
We note that many of the conserved cysteine residues on the
surface of p53, Cys176, Cys242, Cys275 and Cys277 lie
within a region of p53 that is electrostatically positively
charged (Fig. 3 ). This is also the region of p53 that interacts
with DNA. Based on our analysis of the crystal structure it
is most plausible that Cys176 and Cys242 could form a
disulfide bond. However, involvement of other cysteine
residues in either intra- or inter-molecular disulfide bond
formation can not be overlooked.
In fact, it was
demonstrated that recombinant p53 purified in the presence
of chelex-treated solution (presumably metal ion-free) was
unable to bind consensus sequence-containing DNA unless
DTT was added (Rainwater et al., 1995). Apparently, zinc is
tightly bound to p53 during this purification procedure.
Interestingly, adding more zinc to the p53 did not enhance
its DNA binding indicating that DTT may increase p53
binding to DNA by generating reduced free sulfhydryl
groups on cysteine residues not responsible for zinc binding.
In this regard, one group has reported that a small percentage
of recombinant p53 could form a p53-p53 dimer that could
be converted to a monomer after addition of DTT (Delphin
et al., 1994).
If p53 redox control plays an important role in its
regulation one would expect that the cysteine residues at the
surface of the protein would be well conserved. An
alignment of p53 amino acid sequences from 23 species
shows that solvent accessible residues cysteine 176, 242 and
277 share nearly 100% identity (Soussi and May, 1996) but
not other solvent accessible cysteine residues. Cysteine
residues 242 and 277 are conserved throughout all 23
species reported while residue 176 is conserved in 22
species.
VII. Potential sites of p53 cysteine
residue oxidation-a structural analysis
In order for p53 cysteine residues to be redox regulated
the sulfur atoms must be accessible to the oxidant.
Structural studies can be used to rule out many potential
cysteine oxidation sites based on solvent accessibility. Very
few cysteine oxidation reactions on cytoplasmic proteins
have been mapped, which might explain why no consensus
sequence has emerged.
To analyze the structural
requirements for protein cysteine oxidation investigators
have treated purified cytoplasmic proteins of known
structures with glutathione disulfide, a compound that forms
a mixed glutathione disulfide with protein cysteine residues
(Thomas et al., 1995). In the case of rat liver carbonic
anhydrase III, the crystal structure of the protein with
conjugated glutathione was solved. For other proteins a twopart process was used to determine the structural
requirements for glutathione conjugation.
First, the
glutathione-cysteine disulfide was mapped. Second, the
mapped cysteine was assessed for solvent accessibility
through analysis of the crystal structure of the unconjugated
protein. In these studies it was determined that the residues
surrounding the susceptible cysteine residues showed no
consensus sequence. The only consistent feature found was
that cysteine residues were located on the surface of the
protein. Structure analysis of Fos and Jun bound to DNA
also indicated that their oxidation-susceptible cysteine
residues are exposed to the surface as well (Chen et al.,
1998). The fact that solvent accessible cysteine residues are
in close proximity to DNA binding residues may explain
why Fos and Jun oxidation prevents them from binding to
DNA.
We analyzed the crystal structure of residues 94-289 of
p53 bound to DNA (Cho et al., 1994) in order to determine
which of the 10 cysteine residues in this domain may be
exposed to solvent (Connolly, 1983). As shown in Fig. 2,
the sulfhydryl groups of Cys124, Cys176, Cys182, Cys229,
Cys242 and Cys277 can theoretically react with small
molecules on the surface of p53 (indicated with an asterisk).
We conducted our analysis assuming that zinc was absent in
this measurement. It is possible that part of the oxidation
reaction involves removal of zinc as was recently shown for
Hsp33 (Jakob et al., 1999).
To determine which p53 cysteine residues may form
intramolecular disulfide linkages the distances between
cysteine sulfur atoms were measured. Among the cysteine
residues exposed to the solvent only Cys176 and Cys242 can
theoretically form a disulfide bond (the interatomic distance
of sulfur atoms is 3.66 Å). Once zinc is removed and Cys
176 and Cys242 become exposed and/or oxidized, either of
them could attack Cys238, which also appears to be a
reasonable candidate to participate in disulfide bond
formation.
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Figure 2. Analysis of p53 (residues 94-289) for potential cysteine residue oxidation (Cho et al., 1994). Backbone protein chain is
in blue and cysteine side chains are in orange (only the bonds connecting the Cα and Cβ and S atoms are represented ). The white sphere
represents a zinc atom. A thin line between the three cysteine S atoms and zinc are shown representing metal-sulfur bonds. Numbers refer
to cysteine residues; the amino terminus and carboxyl terminus are represented by the 'N' and the 'C', respectively. The solvent exposed
residues are Cys124 , Cys176, Cys182, Cys229, Cys242 and Cys277 (denoted by *). Cys176 and Cys242 can potentially form a disulfide
bond (denoted by **). The structure was displayed and analyzed using the Insight II software, version 98.0 (Molecular Simulations
Incorporated).
predicted to be similar to p53, p73 and p51/p63 (Kaghad et
al., 1997; Osada et al., 1998; Yang et al., 1998). The nearby
double positive charge on residues in close proximity to
Cys176 may render this residue more susceptible to reaction
with a negatively charged oxidizing molecule such as
glutathione disulfide (Snyder et al., 1981). If one or more of
these four cysteine residues is oxidized in p53 it is possible
that oxidation may also alter the activity of p73 and p51/p63.
Only in one species (Ovis aries, sheep) does the p53
sequence reveal a change in Cys176 to Ser176 (Dequiedt et
al., 1995). This is extremely interesting because a serine at
this site is expected to prevent consensus DNA binding
(Rainwater et al., 1995). Other conserved cysteine residues
are maintained in sheep p53. Fig. 4 shows a sequence
alignment between human p53 and squid p53 (the most
divergent of the p53 genes overall). We also show an
alignment of the p53 cysteine coding region with two genes
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Wu et al: Redox modulation of p53
Figure 3. Surface electrostatic potential of p53. The crystal structure shown in Fig. 2 was analyzed for electrostatic potential using
the MolMol program, version 2.6 (Koradi et al., 1996). Blue color represents atoms with low electron density, red color represents residues
with high electron density and white represents neutral and charged residues. The arrows point to positions of the surface exposed sulfur
atoms of the cysteine residues on the surface in this orientation. Note that the orientation of the p53 protein is identical to the p53 structure
shown in Fig. 2. The sulfur atom of the Cys residue 176 was not exposed on this face of the protein.
Figure 4. Sequence comparison of well-conserved solvent exposed p53 cysteine residues. Cys residues expected to be solvent accessible
are in boldface. Conserved charged residues within close proximity to the cysteine residues are also shown at the appropriate polarity and
location.
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Gene Therapy and Molecular Biology Vol 4, page 127
Our structure analysis is based on the crystal structure
of the p53 DNA binding domain bound to a p53-consensus
sequence within an oligonucleotide (the B monomer, Cho et
al., 1994). Although this structure is helpful in providing
predictions of possible oxidation sites it may not provide the
true picture of the conformations p53 exhibits in the cell.
Several reports have shown that p53 is conformationally
flexible and, depending on temperature and oligomeric
status, it can also exist in different conformations in vitro
(Ponchel and Milner, 1998; McLure and Lee, 1999). Within
the crystal structure of p53 bound to DNA there are actually
three monomers of p53 in the repeating subunit (named A, B
and C).
When the images of the monomers were
superimposed differences in some of the cysteine residue
side chain orientations were observed (data not shown).
Thus, it is possible that the conformational changes of p53
observed in the cytosol could expose different cysteine
sulfhydryl groups at the protein surface.
IX. Metal binding agents may alter p53
redox level
A complicating factor in the field of redox chemistry is
the fact that metal ions can catalyze the production of
hydroxyl radicals. Thus, when studying redox changes, free
transition metals (elements in groups IB, IIB, VIB, VIIB and
VII in the periodic table) must always be taken into
consideration.
The biological systems have evolved
molecules to bind free metals but the mechanisms by which
metals are released by these systems are not clear. Metal
ions, in combination with hydrogen peroxide, can form
hydroxyl radicals through the Fenton reaction, also known as
the metal-catalyzed Haber-Weiss reaction:
.
Fe(II) + H2O2 → Fe(III) + OH + OH
(1)
.-
Fe(III) + O 2
→
Fe(II) + O 2
______________________________
..
H2O2 + O2 →
OH + OH + O2
VIII. Mutational analysis of p53
cysteine residues
(2)
(3)
The hydroxyl radical is an extremely reactive species.
It reacts with most substances with diffusion-limiting rate
constants (109- 1010 M-1 s -1). Such reactivity implies a very
short half-life and the molecule will likely be unable to
travel at great distances. Hainaut has investigated the
possibility that copper can modulate p53 DNA binding
properties (Hainaut and Milner, 1993; Hainaut et al., 1995).
Recombinant p53 was translated in rabbit reticulocyte lysate
and exposed to Cu(II) sulfate at 30 µM. Copper exposure
induced wild-type p53 to adopt a denatured conformation as
detected by conformation-dependent antibodies. When
Cu(II) was added to purified recombinant p53 there was no
change in the electron spin resonance (ESR) spectrum
indicating that Cu(II) may not bind to p53. However, when
H2O2 was added to the Cu(II)/p53 mixture, a novel ESR
signal, assigned to Cu(II), was observed indicating binding
of Cu(II) to p53 under these conditions. Hainaut interpreted
these data as follows: the added Cu(II) is initially reduced to
Cu(I) by p53 cysteine residues thereby remaining ESR
silent. After reduction, the Cu(I) is oxidized by H2O2 to
Cu(II) but remains bound to p53. In support of Hainaut's
model, the Cu(I) chelating agent, bathocuproinedisulfonic
acid (BCS) protected the p53-DNA binding activity, an
activity usually inhibited by Cu(II). DMSO, a hydroxyl
radical scavenger, and sodium azide, a singlet oxygen
scavenger, failed to prevent Cu(II)'s ability to inhibit p53's
DNA binding activity ruling out the possibility that these
two oxygen species are potential mediators of p53 oxidation.
Can copper mediate p53 oxidation in cultured cells?
To test the idea that copper may mediate changes in p53
protein properties in vivo the cell permeable copper chelating
agent, pyrrolidine dithiocarbamate (PDTC), was employed
(Nobel et al., 1995; Verhaegh et al., 1997). This agent is
similar in structure to dithiocarbamate-based herbicides,
insecticides and fungicides commonly used in the pesticide
industry (WHO, 1988). Treatment of cells with PDTC led to
inhibition of p53 DNA binding activity and inhibition of
stressor-mediated activation of p21 (presumably via p53
Based on the fact that oxidizing agents prevent p53
binding to consensus-sequence containing DNA and that
some cysteine residues are well-conserved it is predicted that
site-directed mutagenesis of these cysteine residues would
alter p53 activities. Mann and coworkers have conducted a
mutational study examining the importance of p53 cysteine
residues in sequence-specific DNA binding, suppression of
cell transformation and p53-mediated transactivation
(Rainwater et al., 1995). A Cys to Ser substitution at each
cysteine residue of murine p53 was created and the
biochemical and biological activities of each individual
cysteine mutant was compared to wild-type p53. For the
sake of convenience, we will adopt the convention of
enumerating the cysteine residues based on the human
sequence.
From the relative activities in DNA binding,
transactivation and transformation suppression, p53 cysteine
residues were categorized into three groups. One group of
cysteines (those at sites 176, 238 and 242) directly interact
with zinc and are essential for DNA binding, transactivation
and transformation activity of p53. A second group of
cysteine residues (those at sites 124, 135, 141 and 275) is
required for transactivation and suppression function. DNA
binding activity of p53 is maintained when cysteine residues
124, 135, 141 and 275 are changed to serine. The third
group (cysteine residues at sites 182 and 277) did not exhibit
any alterations of the measured activities of p53 when the
residues were changed to serine.
A cysteine to serine substitution is a very conservative
change. An oxidation reaction resulting in a disulfide
linkage may have a more dramatic consequences than a
serine residue substitution. In this regard, it was shown that
Cys to Ser substitution at a redox-sensitive Cys residue in cFos resulted in DTT-independent DNA binding (Okuno et
al., 1993). Thus, oxidation at any of the three classes of
cysteine residues may alter p53 activities.
127
Wu et al: Redox modulation of p53
activation). Interestingly, PDTC-treated nuclear extracts
were incapable of binding to the p53 consensus sequence
DNA. The investigators went on to demonstrate that
intracellular copper levels significantly increased in the
presence of PDTC (Verhaegh et al., 1997). Wu and
Momand (1998) demonstrated that PDTC treatment of
cultured cells led to increased oxidation of p53 protein.
Oxidation of p53 correlated with inhibition of p53-mediated
transactivation, inhibition of p53 nuclear accumulation and
inhibition of UV-induced p53 protein level elevation in
fibroblasts. Interestingly, PDTC treatment also prevented
E6-mediated degradation of p53. These results suggested
that PDTC-mediated copper loading into cells may directly
oxidize p53 or generate hydroxyl radicals near the surface of
p53, resulting in cysteine residue oxidation.
We have incorporated the experimental data into a
coherent testable mechanism for p53 oxidation. Because a
high level of reduced glutathione is present in the cytosol we
have included glutathione as a transporter of electron
radicals.
This reaction mechanism is based on a
combination of previous studies conducted, primarily, on
oxidation of cysteine (Gerweck et al., 1984; Winterbourn,
1993; Thomas et al., 1995):
PDTC-Cu 2+ transport across cell membrane
p53-SH + Cu2+ → p53-S. + Cu1+ + H+
p53-S. + GSH
→
(p53-S-S-G).- + H
+
is required for p53-mediated consensus-sequence binding
(Pavletich et al., 1993; Rainwater et al., 1995; Verhaegh et
al., 1998) Evidence in support of chelation of zinc leading
to p53 conformational changes in cultured cells has recently
been demonstrated with a membrane permeable zinc-specific
chelator
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (Verhaegh et al., 1998). Nuclear
extracts from TPEN-treated cells do not bind consensus
DNA. However, if zinc is supplemented into the TPENtreated media prior to cell harvesting p53 retains the ability
to bind DNA. TPEN alters the conformation of p53 protein.
Importantly, TPEN does not affect the DNA binding activity
of Oct-1, another transcription factor that does not require
zinc. The data suggests that metal ions can modulate p53
transactivation activity, prevent consensus DNA binding by
p53, and can alter the p53 protein conformation. Whether
zinc chelation by TPEN leads to p53 oxidation is unclear at
the moment.
X. Other functions of p53 that may be
regulated by redox levels
Several studies indicate that p53 oxidation prevents its
ability to bind consensus sequence containing DNA. This
leads to the possibility that oxidation may be a mechanism of
p53 down-regulation during oxidative stress. Oxidation of
p53 may constitute a mechanism to turn off p53-mediated
transactivation after its checkpoint function has been
fulfilled and DNA has been repaired. It is also possible that
oxidation regulates p53 function in a positive aspect. Aside
from its role as a transcription factor, biochemical and
genetic studies have demonstrated that p53 is responsible for
the faithful execution of other activities directly related to
maintenance of genome stability. Such activities include
global genomic nucleotide excision repair (Ford and
Hanawalt, 1995; Ford and Hanawalt, 1997; Ford et al., 1998)
and inhibition of homologous recombination (Mekeel et al.,
1997; Dudenhoffer et al., 1998). The p53 activities related
to repair and recombination may result, in part, from direct
interaction of p53 protein with DNA. Some DNA binding
studies suggest that p53 has a direct role in DNA repair. For
example, p53 binds to insertion/deletion mismatches (Lee et
al., 1995; Szak and Pietenpol, 1999), single stranded DNA
molecules, and can mediate DNA strand exchange reactions
(Bakalkin et al., 1994, 1995; Reed et al., 1995; Wu et al.,
1995).
The idea that oxidized p53 may bind non-consensus
DNA and mediate a function has not been extensively
explored. Insertion/deletion mismatch DNA mutations may
result from polymerase-induced errors during replication.
Mann and coworkers have shown that recombinant p53 can
bind to insertion/deletion mismatch DNA in the presence or
absence of DTT (Parks et al., 1997). This may indicate that
this particular DNA binding function of p53 may be
preserved under oxidizing conditions. Furthermore, p53 has
recently been demonstrated to possess double-strand DNA
exonuclease activity (Mummenbrauer et al., 1996; Janus et
al., 1999), which may also be related to a repair function.
The p53 protein has been shown to bind substrates that
(1)
(2)
(3)
(p53-S-S-G) .- + O2
p53-S-S-G + O2.(4)
O2.- + 1/2H2O → 1/2O2 + H2O2 (catalyzed by SOD) (5)
H2O2
→
H2O + 1/2O2
(catalyzed by catalase)
(6)
__________________________________________
p53-SH + Cu2+ + GSH → p53-S-S-G + Cu 1+ + 2H+
According to the reaction mechanism, PDTC transports
Cu 2+ into the cell (Reaction 1). The p53 cysteine residues
are directly oxidized by p53 bound cupric ion (Reaction 2).
After one-electron oxidation of p53 the p53 cysteine residue
carries a thiyl radical that rapidly reacts with free glutathione
(GSH) to form the disulfide radical (Reaction 3). This
radical is very unstable and requires oxidation before it
reforms the reactants. Molecular oxygen oxidizes the radical
to form the S-glutathiolated form of the p53 cysteine residue
(Reaction 4). The superoxide formed by one-electron
reduction of oxygen reacts with water to form hydrogen
peroxide by superoxide dismutase (Reaction 5). Hydrogen
peroxide is then dissociated into water and molecular oxygen
by catalase (Reaction 6). In the future, it will be important
to test this reaction mechanism in vitro.
While free metal ions may lead to p53 oxidation it is
also possible that PDTC indirectly leads to p53 oxidation by
chelating the zinc atom that is bound to Cys176, Cys238 and
Cys242. After zinc removal the p53 cysteine residues may
be oxidized by another molecule, possibly hydroxyl radical.
Both, in vitro and in vivo evidence support the idea that zinc
128
Gene Therapy and Molecular Biology Vol 4, page 129
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mimic recombination intermediates including three stranded
DNA molecules (Dudenhoffer et al., 1998) and Holliday
junctions (Lee et al., 1997). Some p53 mutations found in
human tumors produce mutant p53 proteins that disrupt
DNA repair and recombination activities. Whether p53
oxidation modulates these different p53 activities is unclear
at the moment.
XI. Conclusion
Redox regulation of p53 activity was proposed in 1993
based on the observation that sequence specific DNA
binding could be inhibited by agents that blocked sulfhydryl
groups (Hainaut and Milner, 1993). However, p53 redox
regulation is still poorly understood. The fact that p53
activation in cultured cells can be promoted by agents that
induce the formation of ROI while the oxidation of p53
protein inhibits its ability to bind consensus sequencecontaining DNA in vitro renders this regulation mechanism
even more intriguing. Two disulfide-reducing proteins, Ref1 and thioredoxin reductase may hold the key to
understanding this regulation. By analyzing the structure of
p53, we found several well-conserved cysteine residues
exposed at the protein surface. Another area of investigation
into p53 redox control is related to its ability to bind metal
ions. Binding of metal ions may directly affect p53 redox
potential either at the zinc binding cysteine residues or at
other cysteine residues on the protein surface. To date, most
evidence suggests that p53 oxidation inhibits p53 activity.
However, in vitro studies show that oxidized p53 retains the
ability to bind insertion/deletion mismatches in DNA
without the addition of DTT. This opens the possibility that
redox regulation can be used as a molecular switch inside the
cell to promote other functions of p53 that are less wellknown (Parks et al., 1997). Future studies on the prevalence
of p53 oxidation in response to stressors and the effects of
p53 protein oxidation on its biochemical activities are clearly
needed to address these issues.
Acknowledgments
J.M. is supported by the City of Hope Cancer Center
and H.-H. Wu is supported by the American Cancer SocietyOncology Project Grant (OPG-9-98).
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